The intent of the proposed research is to test a new hypothesis for the way in which neutrophils might function to control infective foci. Although they are thought to act primarily by killing pathogenic microorganisms, these cells also contain a substance in their cytoplasm (not the granules) which can inhibit the growth of a variety of pathogenic microorganisms without killing them. For Candida albicans, at least, the majority of the growth-inhibitory activity of whole neutrophil lysates can be accounted for by this substance. Several lines of evidence indicate the latter to be a calcium binding protein that represents about 30% of the soluble protein of whole neutrophil lysates, and which has previously been identified as a prominent leucocyte antigen termed the cystic fibrosis antigen or the Ll antigen. In view of its newly-described antimicrobial activity, the investigators who originally discovered the Ll antigen have renamed it "calprotectin." Abscess fluids produced experimentally in mice, or obtained as clinical specimens from humans, also contain potent growth inhibitory activity against Candida. This activity, as well as that of the neutrophil cytoplasmic antimicrobial protein, is completely reversible upon the addition of micromolar quantities of zinc ions to the media. The protein from neutrophil cytoplasm appears to accumulate in large amounts in abscess fluids, as demonstrated by SDS-PAGE and western blotting. This substance also appears to directly bind zinc, a characteristic of a number of other calcium binding proteins. Because neutrophils have high turnover rates and very short lifespans at sites of tissue infection, release of this protein after the death of these cells could control the growth of remaining pathogenic microorganisms by depriving them of zinc. If so, then this process may represent a previously undescribed form of host defense. The specific aims of the present proposal are as follows: (1) To purify and characterize the abundant calcium/zinc binding antimicrobial protein of neutrophil cytoplasm; (2) To evaluate the metal-binding properties of the neutrophil cytoplasmic antimicrobial protein; and (3) To study zinc deprivation as an antimicrobial mechanism. The proposed studies will use standard protein isolation techniques to prepare the calcium/zinc binding protein and its individual chains in purified form. These preparations will then be tested for antimicrobial activity, metal binding properties, and their ability to adhere to the surface of the organisms. Finally, the role of zinc binding as an antimicrobial mechanism will be examined in vitro by a dialysis method and by using zinc-deprived organisms, and in the rat air pouch granuloma model of experimental abscesses.